Project description:The optic nerve transfers visual information from the retina to the brain through the axons of retinal ganglion cells (RGCs). In adult mammals, optic nerve injuries and progressive degenerative diseases lead to the irreversible loss of RGCs, resulting in vision loss and blindness. Optogenetic models have proved useful in manipulating the growth of RGCs through expression and stimulation of channelrhodopsins (Chr2) in RGCs using the RGC-specific thy-1 promoter. Using transgenic Chr2 mouse (Thy1-ChR2-EYFP) as a model of regeneration, we profile the lipid changes which occur after traumatic optic nerve crush, light stimulation and forced RGC axonal growth. Thy1-ChR2-EYFP and control (C57BL/6) mice were divided in four groups each - 1) no crush and no stimulation, 2) no crush with stimulation, 3) crush and without stimulation, and 4) crush with stimulation. After euthanasia, the optic nerves were collected for lipidomic analysis. The Bligh and Dyer method was used for lipid extraction, followed by mass spectrometry lipid profiling with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS). The raw scans were analysed with LipidSearch 4.1.3 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001381: [https://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST001381&StudyType=MS&ResultType=5].
Project description:We present lipid profiling data from mouse retina and optic nerve after optic nerve crush and during Wnt3a-induced axonal regeneration at 7 and 15 days post-crush. This data is available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000718).
Project description:In adult mammals, retinal ganglion cells (RGCs) fail to regenerate following damage. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; this can lead to permanent vision loss and, eventually, blindness. Lipids are crucial for the development and maintenance of cell membranes, myelin sheaths, and cellular signaling pathways, however, little is known about their role in axon injury and repair. Studies examining changes to the lipidome during optic nerve (ON) regeneration could greatly inform treatment strategies, yet these are largely lacking. Experimental animal models of ON regeneration have facilitated the exploration of the molecular determinants that affect RGC axon regeneration. Here, we analyzed lipid profiles of the ON and retina in an ON crush rat model using liquid chromatography-mass spectrometry. Furthermore, we investigated lipidome changes after ON crush followed by intravitreal treatment with Zymosan, a yeast cell wall derivative known to enhance RGC regeneration. This data is available at the NIH Common Fund's Metabolomics Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, the Metabolomics Workbench, http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53.
Project description:The optic nerve is part of the mammalian adult central nervous system (CNS) and has limited capability to regenerate after injury. Deletion of phosphatase and tensin homolog (PTEN), a negative regulator of the PI3 kinase/Akt pathway, has been shown to promote regeneration in retinal ganglion cells (RGCs) after optic nerve injury [1]. We present the lipidome of adult PTENloxP/loxP mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of CNS neuroregeneration. At 4 weeks old, PTENloxP/loxP mice were intravitreally-injected with 2-3 μl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated time-points after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. A modified Bligh and Dyer [2] method was used for lipid extraction from the optic nerves, followed by liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Accela 600 HPLC. The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001477.
Project description:Zebrafish (Danio rerio) have the capacity for successful adult optic nerve regeneration. In contrast, mammals lack this intrinsic ability and undergo irreversible neurodegeneration seen in glaucoma and other optic neuropathies. Optic nerve regeneration is often studied using optic nerve crush, a mechanical neurodegenerative model. Untargeted metabolomic studies within successful regenerative models are deficient. Evaluation of tissue metabolomic changes in active zebrafish optic nerve regeneration can elucidate prioritized metabolite pathways that can be targeted in mammalian systems for therapeutic development. Female and male (6 month to 1 year old wild type) right zebrafish optic nerves were crushed and collected three days after. Contralateral, uninjured optic nerves were collected as controls. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient metabolite concentrations for analysis. Optic nerve regeneration at 3 days post crush was demonstrated by microscope visualization of GFP fluorescence in Tg(gap43:GFP) transgenic fish. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites standards.
Project description:The right optic nerve of adult, 6 month to 1 year old, female and male Danio rerio were crushed and collected three days after. Matching controls of uninjured left optic nerves were also collected. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) n = 24 to obtain sufficient tissue for analysis. The brain from one male fish was also collected for control/calibration. Lipid extraction was done with the Bligh and Dyer [1] method, followed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. The lipids were identified and quantified with LipidSearch 4.2.21 and the statistical analysis was conducted through Metaboanalyst 5.0. This data is available at Metabolomics Workbench, Study ID ST001725.
Project description:Optic nerve (ON) regeneration in mammalian systems is limited by an overshadowing dominance of inhibitory factors. This has severely hampered the identification of pro-regenerative pathways. Here, we take advantage of the regenerative capacity of larval zebrafish to identify pathways that promote ON regeneration. From a small molecule screen, we identified modulators of serotonin (5-HT) signaling that inhibit ON regeneration. We find several serotonin type-1 receptor genes are expressed in RGC neurons during regeneration and that inhibiting 5-HT1 receptors or components of the 5-HT pathway selectively impedes ON regeneration. We show that 5-HT1 receptor signaling is dispensable during ON development yet is critical for regenerating axons to emerge from the injury site. Blocking 5-HT receptors once ON axons have crossed the chiasm does not inhibit regeneration, suggesting a selective role for 5-HT receptor signaling early during ON regeneration. Finally, we show that agonist-mediated activation of 5-HT1 receptors leads to enhanced and ectopic axonal regrowth. Combined, our results provide evidence for mechanisms through which serotonin-dependent neuromodulation directs ON regeneration in vivo.